Equine herpesvirus vaccine

ABSTRACT

A vaccine for protecting a horse against diseases associated with EHV-1 and/or EHV-4 is provided. The vaccine commonly includes inactivated EHV-1 (e.g., chemically inactivated EHV-1 KyA virus) and an adjuvant. The adjuvant can include a cross-linked olefinically unsaturated carboxylic acid polymer which may have bioadhesive properties. The vaccine may also include antigens against other equine pathogens such as inactivated EHV-4 and inactivated A1 and/or A2 strains of equine influenza virus. Methods for protecting horses against diseases associated with EHV-1 and/or EHV-4 and methods of producing the equine herpesvirus vaccine are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/897,984, filed Jul. 23, 2004, which is a continuation of U.S.application Ser. No. 09/812,720, filed Mar. 20, 2001 (issued as U.S.Pat. No. 6,803,041), the disclosures of which are incorporated herein byreference in their entirety.

BACKGROUND

Respiratory diseases are a major cause of economic loss to the equineindustry. Equine herpesviruses (EHV), equine influenza viruses (EIV),and the bacterium, Streptococcus equi are pathogens most oftenassociated with infectious respiratory disease in horses. World wide,equine herpesviruses are major pathogens associated with morbidity inhorses as a result of respiratory infection. Both equine herpesvirustype 1 (EHV-1) and type 4 (EHV-4) can cause respiratory disease. EHV-1is also associated with abortions and neurological disease. Because ofthe high degree of mobility and the international nature of the equineindustry, efficacious vaccines are needed to reduce the disease andcontrol the spread of these pathogens.

A number of EHV vaccines are available commercially. None, however,generally is capable of conferring long lasting protection and mostrequire frequent booster immunizations to achieve a significant level ofprotection against EHV infection. The most commonly recommended route ofadministration is via intramuscular injection, despite the respiratorysystem being a primary site of the infection in many instances. Inaddition, some of the commercial vaccines have been reported to causeundesirable side effects. A number of attempts at developing arecombinant vaccine for EHV have been reported. This approach, however,has not yet resulted in the introduction of a commercial recombinantvaccine which has achieved widespread acceptance.

Literature reports have consistently documented a high degree ofvariability in the capability of vaccines based on EHV-1 strains toprovide cross protection against infection by EHV-4 strains. Whilevaccines based on EHV-4 strains have shown a greater propensity toprovide some protection against both EHV-1 and EHV-4 strains, crossprotection based on EHV-4 strains has also been reported to showvariability.

There is accordingly a continuing need to develop additional vaccinescapable of protecting horses against diseases associated with EHV-1and/or EHV-4. It would also be advantageous to develop vaccine that iseffective against EHV-1 and/or EHV-4 which could be administered viaintranasally as well as via parenteral methods (e.g., intramuscularly,subcutaneously or intravenously).

SUMMARY

The present invention relates to immunogenic compositions which includean inactivated form of EHV-1. In particular, the application provides avaccine for protecting horses against diseases associated with EHV-1and/or EHV-4. The vaccine includes inactivated EHV-1 (e.g., chemicallyinactivated EHV-1 KyA virus) and typically also includes an adjuvant.The vaccine may also include other components, such as preservative(s),stabilizer(s) and antigens against other equine pathogens. Typically,the antigens against other equine pathogens are also present in aninactivated form, such as inactivated forms of EHV-4 and inactivatedstrains of equine influenza virus (“EIV”). For example, the vaccine maybe a combination vaccine which includes inactivated forms of A1 and/orA2 strains of equine influenza virus in addition to the inactivatedEHV-1. Examples of suitable antigens against EIV include inactivated EIVA1 virus strain A/EQ1/Newmarket/77, inactivated EIV A2 virus strainNewmarket/2/93, and inactivated EIV A2 virus strain Kentucky/95.

The terms “vaccine” and “immunogenic composition” are defined herein ina broad sense to refer to any type of biological agent in anadministratable form capable of stimulating an immune response in ananimal inoculated with the vaccine. For purposes of this invention, thevaccine (immunogenic composition) typically includes the viral agent inan inactivated form. Vaccines in general may be based on either thevirus itself or an immunogenic (antigenic) component of the virus.Herein, the term “protection” when used in reference to a vaccine refersto the amelioration (either partial or complete) of any of the symptomsassociated with the disease or condition in question. Thus, protectionof horses from EHV by the present vaccines generally results in adiminishing of virus shedding and/or one or more of the clinicalsymptoms associated with infection by EHV-1 and/or EHV-4 (e.g., pyrexia,nasal discharge, conjunctivitis, coughing, dyspnea, depression, andantibiotic treatment required for secondary bacterial infection).

In one embodiment, the present immunogenic compositions include achemically inactivated form of EHV-1. Vaccines which include chemicallyinactivated EHV-1 KyA virus are particularly desirable. A variety ofchemical inactivating agents known to those skilled in the art may beemployed to inactivate the virus. Ethylenimine and related derivatives,such as binary ethylenimine (“BEI”) and acetylethylenimine, are examplesof suitable chemical inactivating agents for use in inactivating theEHV-1 virus. Other chemical inactivating agents, e.g.,beta-propiolactone or aldehydes (such as formaldehyde andglutaraldehyde), can also be used to inactivate the virus.

The present vaccines generally include an adjuvant which desirably mayhave bioadhesive properties, particularly where the virus is designed tobe capable of intranasal administration. Examples of suitable adjuvantsinclude cross-linked olefinically unsaturated carboxylic acid polymers,such as cross-linked acrylic acid polymers. As used herein the term“cross-linked acrylic acid polymer” refers to polymer and copolymersformed from a monomer mixture which includes acrylic acid as thepredominant monomer in the mixture. Examples of suitable cross-linkedacrylic acid polymers include those commercially available under thetradenames Carbopol® 934P and Carbopol® 971 (available from B.F.GoodrichCo., Cleveland, Ohio). One particularly suitable adjuvant for use in thepresent vaccines is a cross-linked acrylic acid polymer having aBrookfield viscosity of no more than about 20,000 cPs (as measured at 20rpm as a 1.0 wt. % aqueous solution at pH 7.5). Where a bioadhesiveadjuvant is desired, it may be advantageous to utilize an adjuvant whichhas a bioadhesive property of at least about 50 dynes/cm² as measuredbetween two pieces of freshly excised rabbit stomach tissue(as.determined by the procedure described in U.S. Pat. No. 4,615,697).

Methods for protecting horses against diseases associated with EHV-1and/or EHV-4 which include administering a vaccine containinginactivated EHV-1 to the horses. The vaccine can be administered using avariety of methods including intranasal and/or parenteral (e.g.,intramuscular) administration. In one embodiment of the method, theinactivated EHV-1 containing vaccine is first administeredintramuscularly one or more times (e.g., at intervals of 2-4 weeks),followed by administration of the vaccine at least once intranasally(e.g., 2-4 weeks after the last parenteral administration of vaccine).The vaccine is advisedly administered to horses that are 6 months orolder. Ideally, all horses in a given herd are Vaccinated annually inorder to protect against the spread of respiratory symptoms of thedisease.

A method of producing an equine herpesvirus vaccine is also provided.The method typically includes inoculating simian cells with EHV-1 virus,e.g., with EHV-1 KyA virus. The inoculated simian cells are incubatedgenerally at least until CPE is observed (commonly after 24 to 120 hoursat 36° C.), and then the EHV-1 virus is harvested from the incubatedcells (e.g., by decanting and filtering the culture fluids). Theharvested virus-containing fluids can be treated with a chemicalinactivating agent, such as binary ethylenimine, to form inactivatedEHV-1 virus. Typically, the inactivated virus is further processed,e.g., by concentration and blending with other components, to produce acommercial formulation. For example, the fluids containing theinactivated virus may be concentrated and blended with an adjuvantand/or antigen(s) to one or more other equine pathogens.

The present application is also directed to a kit which includes incombination, (1) a dispenser capable of administering a vaccine to ahorse; and (2) a chemically inactivated EHV-1 containing vaccine capableof protecting against diseases associated with EHV-1 and/or EHV-4. Thekit may include a dispenser which is capable of dispensing its contentsas droplets, e.g., as aerosol, atomized spray and/or liquid droplets,and a form of the vaccine which is capable of protecting againstdiseases associated with EHV-1 and/or EHV-4 when administered at leastin part intranasally.

Throughout this application, the text refers to various embodiments ofthe present compositions and/or related methods. The various embodimentsdescribed are meant to provide a variety of illustrative examples andshould not be construed as descriptions of alternative species. Rather,it should be noted that the descriptions of various embodiments providedherein may be of overlapping scope. The embodiments discussed herein aremerely illustrative and are not meant to limit the scope of the presentinvention.

DETAILED DESCRIPTION

The present immunogenic compositions include an inactivated form ofEHV-1. The vaccines are designed for protecting horses against diseasesassociated with EHV-1 and/or EHV-4. The vaccines typically include achemically inactivated form of EHV-1 and those which include chemicallyinactivated EHV-1 KyA virus are particularly desirable. A variety ofchemical inactivating agents known to those skilled in the art may beemployed to inactivate the virus. Ethylenimine and related derivatives,such as binary ethylenimine (“BEI”) and acetylethylenimine, are examplesof suitable chemical inactivating agents for use in inactivating theEHV-1 virus. Other chemical inactivating agents, e.g.,beta-propiolactone, aldehydes (such as formaldehyde) and/or detergents(e.g., Tween® detergent, Triton® X, or alkyl trimethylammonium salts)can also be used to inactivate the virus. The inactivation can beperformed using standard methods known to those of skill in the art.Samples can be taken at periodic time intervals and assayed for residuallive virus. Monitoring of cytopathic effect on an appropriate cell lineand/or fluorescent staining with an appropriate specific monoclonalantibody can be used to detected the presence of residual live virus.

Inactivation with BEI can be accomplished by combining a stock BEIsolution (e.g., a solution formed by adding 0.1-0.2 M 2-bromo-ethylaminehydrobromide to 0.1-0.2 N aqueous NaOH) with viral fluids to a finalconcentration of about 1-2 mM BEI. Inactivation is commonly performed byholding the BEI-virus mixture at 35-40° C. (e.g., 37° C.) with constantmixing for 36-72 hours. Virus inactivation can be halted by the additionof sodium thiosulfate solution to a final concentration in excess of theBEI concentration (e.g., 2-3 mM sodium thiosulfate with 1-2 mM BEIsolutions) followed by mixing for several hours.

The present immunogenic compositions usually include an adjuvant and, ifdesired, one or more emulsifiers such as Tween® detergent incorporatedwith the inactivated EHV-1. Suitable adjuvants include, for example,vitamin E acetate solubilisate, aluminum hydroxide, aluminum phosphateor aluminum oxide, (mineral) oil emulsions, non-ionic detergents,squalene and saponins. Other adjuvants which may be used include an oilbased adjuvants such as Freund's complete adjuvant (FCA), and Freund'sincomplete adjuvant (FIA). It has been found that cross-linkedolefinically unsaturated carboxylic acid polymers, such as Carbopol® 971polymer, are particularly suitable adjuvants for use in the presentinactivated EHV-1 immunogenic compositions.

One example of such an adjuvant is an olefinically unsaturatedcarboxylic acid polymer produced by reaction of a monomer mixture whichincludes one or more olefinically unsaturated carboxylic acid monomers(such as acrylic acid and/or methacrylic acid) and a cross-linkingagent. Typically, at least about 90 wt. % of the monomer mixture isolefinically unsaturated carboxylic monomer. The resulting polymerproduct desirably contains no more than about 0.5 wt. % and, preferably,no more than about 0.2 wt. % unreacted olefinically unsaturatedcarboxylic monomer. The polymerization reaction can be carried out byreaction of the monomer mixture in the presence of solvent whichincludes aliphatic ketone, alkyl ester or a mixture thereof. Suitablealiphatic ketones include those having 3 to 6 carbon atoms, such asacetone and cyclohexanone (as used herein the term “aliphatic ketone”includes cycloaliphatic ketones). Examples of suitable alkyl estersinclude those having 3 to 6 carbon atoms, such as ethyl acetate, ethylformate, isopropyl acetate, n-propyl acetate, butyl acetate or a mixturethereof.

Suitable olefinically unsaturated carboxylic acid polymer adjuvantsdesirably have a Brookfield viscosity of no more than about 40,000 cPs(at 20 rpm as a 0.5 wt. % aqueous solution at pH 7.5). Particularlysuitable examples include olefinically unsaturated carboxylic acidpolymers with a viscosity of no more than about 15,000 cPs and moredesirably about 4,000-11,000 cPs (at 20 rpm as a 0.5 wt. % aqueoussolution at pH 7.5).

One example of a suitable adjuvant includes a cross-linked acrylic acidpolymer formed from a monomer mixture which includes acrylic acid and across-linking agent. The cross-linking agent may include a polyalkenylpolyether cross-linking agent, such as a divinyl glycol. Examples ofsuitable divinyl alcohols include allyl sucrose, allyl pentaerythritol,polyalkylene diol diallyl ether having a molecular weight of no morethan 1000, trimethylolpropane diallyl ether, and mixtures thereof.Examples of other useful cross-linking agents are divinylbenzene,N,N-diallylacrylamide, 3,4-dihydroxy-1,5-hexadiene,2,5-dimethyl-1,5-hexadiene and the like.

Where the vaccine is to be administered intranasally, it may beadvantageous to use an adjuvant is bioadhesive with respect to mucousmembranes. Bioadhesive polymers generally have the property of beingable to adhere to a mucous membrane in the eyes, nose, mouth,gastrointestinal tract, vaginal cavity and rectal canal. Bioadhesive maybe broadly defined as a material that adheres to a live or freshlykilled biological surface such as mucus membrane or skin tissue.Bioadhesion as that phrase is used herein to define a useful bioadhesiveis assayed by a procedure that measures the force required to separatetwo layers of freshly excised rabbit stomach tissue that are adheredtogether by an adhesive. Using this procedure, a bioadhesive may bedefined as a material that requires a force of at least about 50dynes/cm.sup.2 to separate two adhered, freshly excised pieces of rabbitstomach tissue, following the procedure described in U.S. Pat. No.4,615,697, the disclosure of which is herein incorporated by reference.The upper limits for forces required to separate the freshly excisedrabbit tissue are not precisely known, but are believed to be at leastabout 2000 dynes/cm^(2.)

Suitable examples of adjuvants include cross-linked olefinicallyunsaturated carboxylic acid polymers with bioadhesive properties (e.g.,Carbopol® 971 polymer, a cross-linked acrylic acid polymer availablefrom B.F.Goodrich Co., Cleveland, Ohio). Polyacrylic acids of this typeare generally crosslinked carboxy-functional polymers that containspecified amounts of carboxyl functionality and crosslinking agent. Suchpolymers can be a bioadhesive such that the polymers exhibit an adhesionbetween two pieces of freshly excised rabbit stomach tissue of at least50 dynes/cm.sup.2 (when measured in the manner described in U.S. Pat.No. 4,615,697).

It is generally advantageous to formulate the present compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to the treated;each unit containing a predetermined quantity of the active materialcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the dosageunit forms of inactivated EHV-1 (as well as inactivated EHV-4 and/orinactivated EIV) are dictated by and depend on among other factors (a)the unique characteristics of the active material and the particulartherapeutic effect to be achieved; (b) the limitations inherent in theart of compounding such active material for the treatment of disease;and (c) the manner of intended administration of the dosage unit form.

The principal active ingredient is typically compounded for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as disclosedherein. A unit dosage form can, for example, contain the EHV-1 antigenin amounts ranging from 1 to about 5 relative potency units (“R PUs”).This amount of the antigen is generally present in from about 1 to about25/ml of carrier. In the case of compositions containing supplementaryactive ingredients (e.g., inactivated EIV and/or inactivated EHV-4), thedosages are determined by reference to the usual dose and manner ofadministration of the supplementary active ingredients.

The present vaccines typically include inactivated EHV-1 formulated witha pharmaceutically acceptable carrier. The pharmaceutical forms suitablefor injectable use commonly include sterile aqueous solutions (wherewater soluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Theformulation should desirably be sterile and fluid to the extent thateasy syringability exists. The dosage form should be stable under theconditions of manufacture and storage and typically is preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example,. glycerol, propyleneglycol, liquid polyethylene glycol, and the like), suitable mixturesthereof and vegetable oils. One possible carrier is a physiological saltsolution. The proper fluidity of the solution can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal(sodium ethylmercuri-thiosalicylate), deomycin, gentamicin and the like.In many cases it may be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions, if desired, can be brought about by the use inthe compositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating theinactivated virus in the desired amount in an appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions can be prepared byincorporating the various active ingredients into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum-drying and the freeze-drying techniquewhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

It may also be advantageous to add a stabilizer to the presentcompositions to improve the stability of inactivated virus. Suitablestabilizers include, for example, glycerol/EDTA, carbohydrates (such assorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose),proteins (such as albumin or casein ) and protein degradation products(e.g., partially hydrolyzed gelatin). If desired, the formulation may bebuffered by methods known in the art, using reagents such as alkalimetal phosphates, e.g., sodium hydrogen phosphate, sodium dihydrogenphosphate, potassium hydrogen phosphate and/or potassium dihydrogenphosphate. Other solvents, such as ethanol or propylene glycol, can beused to increase solubility of ingredients in the vaccine formulationand/or the stability of the solution. Further additives which can beused in the present formulation include conventional antioxidants andconventional chelating agents, such as ethylenediamine tetraacetic acid(EDTA).

The compositions and methods of the present invention may be illustratedby the following examples, which are presented to illustrate the presentinvention and to assist in teaching one of ordinary skill how to makeand use the same. These examples are not intended in any way to narrowor otherwise limit the scope of the present invention.

EXAMPLE 1 Production of the Fluids Containing Inactivated EHV-1 Strain

To produce the Equine Rhinopneumonitis Vaccine, killed virus, a masterseed culture of an EHV-1 was first produced. From this master seed, aculture of EHV-1 was grown and then inactivated. The inactivated virusculture was then mixed with an adjuvant in order to produce the EquineRhinopneumonitis Vaccine. The following method was used to produce theEquine Rhinopneumonitis Vaccine. In order to produce the EHV-1 masterseed virus culture (“EHV-1 MSV”), equine herpesvirus type 1 strain KyA(EHV-1 KyA) was passaged four times on Vero A139 cells and four times onEVero cells. The fourth passage was used as a master seed virusdesignated EHV-1 KyA, MSV Lot 001-dil.

From the master seed virus, a culture of EHV-1 was produced by infectingEVero cells with EHV-1 MSV in a minimum essential medium (“MEM”) having0 to 5% serum. Gentamicin was added to the culture medium in an amountsufficient to inhibit bacterial growth. The EVero cells were typicallyinfected with the EHV-1 MSV with a target multiplicity of infection (“MOI”) of 0.001. Such cultures can be grown in glass roller bottles or onmicrocarrier beads. The culture was incubated at 36° C.±2° C. for 24 to120 hours until cytopathic effect (“CPE”) was observed. Duringincubation, the culture was monitored for EHV induced CPE to ensure apure EHV strain. If atypical CPE was observed or any macroscopic ormicroscopic evidence of contamination existed, the culture wasdiscarded. Pure virus culture was aseptically harvested into sterileglass carboys, sterile plastic carboys, or sterile stainless steel tanksand was clarified by filtration through filters of 8 microns or greater.Bulk virus harvest fluids were tested to ensure the absence ofmycoplasma prior to inactivation. Harvested fluids which were notimmediately inactivated were stored at −40° C. or below.

After being harvested, the virus culture was inactivated in order toproduce a killed vaccine. To inactivate the virus, the culturetemperature was brought to 36° C.±2° C. Next, a 0.2M solution of2-bromoethyleneamine hydrobromide was cyclized to binary ethylenimine(“BEI”) in 0.15M NaOH and added to the culture to give a finalconcentration of 2 mM BEI. The resulting mixture was stirredcontinuously for 48 hours at 36° C.±2° C.

After treatment with BEI, the culture was tested for its ability toinduce CPE typical of EHV to ensure inactivation of the virus using theprocedure described in Example 3. This task was accomplished by passingthe BEI treated viral fluids over EVero cells and checking the EVerocells for any viral infection. The BEI treated culture fluids weretypically stored at 2-7° C. or below until the inactivation assay hadbeen completed. After a satisfactory inactivation test showing no viralinfection, excess BEI was neutralized by adding a sufficient amount of acold (4° C.±2° C.) solution of 1.0 M sodium thiosulfate to give a finalconcentration of 6 mM.

Following the inactivation and testing of the EHV-1 culture, theinactivated culture was concentrated, if necessary, by ultrafiltrationto a concentration that would allow formulation as a vaccine with arelative potency (“R P”) for EHV-1 of at least 1.0 as determined by theEHV potency release assay described in Example 6 herein.

The inactivated virus was formulated as an adjuvanted vaccine bythoroughly blending the inactivated EHV-1 culture with saline and a 0.5%stock solution of the adjuvant Carbopol® 971 to form a bulk serial. Atypical 60 L serial was made by blending 3.5-4.0 L inactivated EHV-1culture fluid, 12 L Carbopol® 971 stock solution and 44-44.5 L saline.The bulk serial was maintained at 2-8° C. until being transferred intovials containing either one or ten doses (@ 2.2 ml per dose). Each doseof the inactivated vaccine contained at least 1.0 RP value ofinactivated EHV-1, 2 mg Carbopol® 971 and a residual amount ofgentamicin.

EXAMPLE 2 Production of the Fluids Containing Inactivated EquineInfluenza Virus

Equine Influenza virus—A/EQ1/Newmarket/77, subtype A1

The MSV Rec ER1K subtype A1 of equine influenza was developed at theWellcome Foundation Ltd., Beckenham, Kent, U.K. The original equinestrain was passaged ten times, alone or in combination with A/PuertoRico/8/34 virus, in specific pathogen free (SPF) embryonated eggs.Reassortant ERIK was passaged an additional seven times in Vero tissueculture to produce MSV designated A/E/Newmarket/77 (Equi) (H7N7) RecERIK. The virus was received by Boehringer Ingelheim Vetmedica, Inc.(BIVI) from Coopers Animal Health, Inc., Est. Lic. No. 107. The viruswas passed one time in the EVero cell line [Vero cell line received fromCoopers Animal Health has been designated as EVero at BIVI] at BIVI toestablish the new master seed virus. The master seed virus is designatedas Lot 111795.

Equine Influenza virus—Newmarket/2/93, subtype A2

The Newmarket/2/93 subtype A2 of equine influenza was obtained from Dr.J. A. Mummford at the Animal Health Trust, P.O. Box 5 Newmarket, SuffolkCB8 8JH, England. The virus was isolated from a horse with rhinitis. Thevirus was passaged five times in specific pathogen free (SPF)embryonated chicken eggs and then passaged five times in the Madin-Darbycanine kidney (MDCK) cell line. The fifth passage in MDCK cells wasdesignated as MSV. The MSV is designated as EIV NM/2/93, MSV Lot001-dil.

Equine Influenza—Kentucky/95, subtype A2

The Kentucky/95 subtype A2 of equine influenza was obtained from theGluck Equine Research Center, Lexington, Ky. The virus was isolated froma horse with rhinitis. The virus was passaged two times in specificpathogen free (SPF) embryonated chicken eggs. The virus was thenpassaged three times in Madin-Darby canine kidney (MDCK) cell line andthree times in EVero cells. The third passage in EVero cells wasdesignated as MSV. The MSV is designated as EIV K95, MSV Lot 001-dil.

The following procedures were used to produce the three strains ofequine influenza components separately, but by similar methods. Eachproduction lot of Newmarket/77 virus was identified as equine influenzavirus (EIV) by observing characteristic EIV induced cytopathic effects(CPE) in EVero cells. Each production lot of Newmarket/2/93 andKentucky/95 virus was identified as EIV by observing characteristic EIVinduced CPE in MDCK cells. The Newmarket/77 and Kentucky/95 arecytocidal for monkey kidney cell cultures and produced typical EIV CPEin monolayer cultures. The Newmarket/2/93 is cytocidal for MDCK cellcultures and produced typical EIV CPE in monolayer cultures. Virulencein horses was not evaluated for any of the viruses. The master seedviruses were tested for purity in accordance with 9 C.F.R. 113.27 (c),113.28, and 113.55. The Newmarket/77 master seed virus was for SwineVesicular Disease, Akabane, Bovine Ephemeral Fever, Bluetongue, andVelogenic Viscerotrophic Newcastle disease. Bulk virus harvest fluidsare tested for mycoplasma prior to inactivation in accordance with the 9C.F.R. 113.28. The master seed viruses were tested for immunogenicityusing the following procedure.

Bulk or final container samples of completed product from each serialwere tested for potency by a guinea pig potency test. Each of at leastten guinea pigs, weighing 300-500 grams, was injected intramuscularly.Each guinea pig dose was one half of the dose recommended as a unit doseof the vaccine on the label for a horse. A second dose was injected 14to 21 days after the first dose. Two additional guinea pigs from thesame source were held as controls. Fourteen to 21 days after the secondinjection, serum samples from each vaccinate and each control weretested for Newmarket/77, Newmarket/2/93 and Kentucky/95 antibodies byhemagglutination inhibition (HAI). The potency of the EIV fractions inthe vaccine were determined using the National Veterinary ServicesLaboratories Testing Protocol, Supplemental Assay Method for Conductingthe Hemagglutination Inhibition Assay for Equine Influenza Antibody(MVSAM0124.01, dated Oct. 2, 1998).

If the controls did not remain seronegative at 1:10 for the EIV fractionunder test, the test was considered a no test and was repeated. If eightof the ten vaccinates in a valid test did not develop HAI antibody titerof 80 or greater for the Newmarket/77 fraction and a titer of 40 orgreater for the Newmarket/2/93 and Kentucky/95 fractions, the sample wasconsidered unsatisfactory. Vaccines containing inactivated EIV aredesirably formulated to include at least about 64 HAU/unit dose ofinactivated EIV virus subtype A1 and/or at least about 256 HAU/unit doseof inactivated EIV virus subtype A2.

The first passage through the fourth passage from master seed virus wereutilized for working and production seed. Vaccine was produced from thefirst passage through the fifth passage from master seed virus. EquineInfluenza, strain Newmarket/77, subtype A1 and Equine Influenza, strainKentucky/95, subtype A2 were propagated in EVero Cell Line Cultures.

The MDCK cell line used for propagation of Newmarket/2/93 was confinedbetween passages 71 and 91. Passage 71 was frozen and was designated asthe Master Cell Stock. The MDCK cell cultures were propagated in MEMcontaining 5-10% fetal bovine serum. Seed and production culture ofNewmarket/2/93 were propagated in MEM supplemented with 10 units oftrypsin/mL. Cell cultures are grown on microcarrier beads in 1, 3, 10,30, 50, or 140 liter cell culture vessels, 9 L glass roller bottles, orplastic roller bottles. Master and working seed viruses are stored at−60° C. or below.

MDCK cells cultures were grown in sterile glass or plastic containers.The cultures were grown in 75 cm² or 150 cm² plastic flasks with 40 to100 mL of media. Expansion cultures were grown in roller bottle culturesin 100-1000 mL media and then subcultured in roller bottle culturesalso. Either a 75 cm² or 150 cm² surface attachment area was chosen,based upon the attachment and growth of the cell line when first comingout of the liquid nitrogen repository. Due to the stress of liquidnitrogen storage, in some instances it was advantageous to start cellgrowth in a smaller surface area, and when growth had been achieved,subculture the cells further into a 150 cm² T-flask. If the cells cameout of liquid nitrogen storage and produced a healthy monolayer of cellssufficient for expansion in a 150 cm² T-flask, then the 75 cm² T-flaskstep was omitted and the cells were planted directly into the 150 cm²T-flask.

Prior to inoculation with virus, the cell growth medium was decantedfrom the culture vessel. Minimum essential medium (MEM) without serumwas added to the culture vessel and the cells were rinsed for 30 to 45minutes. The volume of rinse medium was dependent on the culture vesselbeing used. The culture vessel was inoculated by decanting rinse mediumand adding MEM without serum containing 10 Units of purified trypsin/mlMEM and to which seed virus has been added. Cell cultures were infectedwith a target multiplicity of infection (MOI) of 0.01 for theNewmarket/77, Newmarket/2/93 and Kentucky/95 strains.

The cell cultures were maintained at 36° C.±2° C. until the fluid washarvested. Monolayers were observed following inoculation forcharacteristic EIV induced changes in cell morphology. CPE includedrounding and refractile cells that detached from the monolayer surface.CPE was usually apparent 24-96 hours after inoculation. The culture wasexamined throughout the incubation period for macroscopic and/ormicroscopic evidence of contamination or for atypical changes in cellmorphology. Any culture exhibiting evidence of microbial contaminationor nonspecific cellular degeneration was discarded.

Prior to harvesting, each culture was examined macroscopically andmicroscopically. Any culture exhibiting evidence of contamination oratypical CPE was discarded. The fluid from the culture vessel(s) washarvested one to four days after inoculation. The virus culture fluid inthe culture vessel was aseptically harvested into sterile glass orplastic carboys or a sterile stainless steel tank after clarification byfiltration through filters of 8 microns or greater. Pooled fluids wereeither inactivated immediately or stored at −40° C. or below forinactivation at a later time. Samples were collected for sterility andantigen testing after inactivation.

The volume of culture fluids was determined and the temperature of thefluids was brought to 36° C.±2° C. A 0.2M solution of2-bromoethyleneamine hydrobromide which had been cyclized to binaryethylenimine (BEI) in 0.15M NaOH was added to the culture fluids to givea final BEI concentration of 2 mM. The inactivated virus fluid wasstored either frozen at ≦−40° C. or at 4-8° C. until completion ofinactivation testing.

Each lot of Newmarket/77 and Kentucky/95 was tested for inactivation bypassage in Vero A139 cells. Each lot of Newmarket/2/93 was tested forinactivation by passage in MDCK cells. Appropriate cell culturemonolayer (150 ml) was inoculated with 1.0 ml of inactivated EIV fluidsand maintained at 36° C.±2° C. for 14 days with at least two passages.At the end of the maintenance period, the cell monolayers were examinedfor CPE typical of EIV. For positive virus controls, one culture flaskeach of EVero cells was inoculated with reference Newmarket/77 andKentucky/95 virus, respectively, and one culture of MDCK cells wasinoculated with a reference Newmarket/2/93 virus (each to a target MOIof 0.01). One flask of EVero cells and one flask of MDCK cells remaineduninoculated as negative controls. After incubation and passage, theabsence of virus-infected cells in the BEI treated viral fluidsconstituted a satisfactory test for inactivation. The control cellsinoculated with the reference virus showed CPE typical of EIV and theuninoculated flask showed no evidence of EIV CPE.

After a satisfactory inactivation test, residual BEI was neutralized bythe addition of a cold (4° C.±2° C.) solution of 1.0 M sodiumthiosulfate to give a final concentration of 6 mM. The inactivatedfluids were stored at 4° C.±2° C. or below until bulking of the finalproduct. If the inactivation test was unsatisfactory, the fluids couldbe retreated with BEI using the procedure described above and retestedfor inactivation.

If necessary, each lot of inactivated bulk virus fluid was concentrated2× to 50× by ultrafiltration to achieve the desired concentration. Oneor more bulk lots of inactivated bulk virus fluids were usually pooledfor concentration. The concentrated bulk virus was held at 4-8° C. untilbulking.

The resulting product after inactivation and concentration containedgentamicin in residual amounts from the medium used in the production ofthe virus harvest fluids. These levels did not exceed the level allowedper dose of product. The vaccine was formulated to contains 2 mg ofCarbopol® 971 per dose of vaccine.

The components for bulking were aseptically added to a glass, plastic,or stainless steel container by siphoning, or by positive pressure(sterile-filtered nitrogen). The serial was blended thoroughly and thenmaintained at 2-8° C. until ready for filling into final containers. Thefollowing provides an illustrative inactivated EIV vaccine composition:Newmarket/77 3,000 ml Newmarket/2/93 6,000 ml Kentucky/95 6,000 mlCarbopol ® 971 (0.5% stock solution) 12,000 ml Saline 33,000 mlIf desired, the saline or a portion thereof can be substituted by asolution that contains inactivated EHV-1 and, optionally, inactivatedEHV-4 to produce a combination EHV/EIV vaccine.

After bulking, the serial was drawn off into sterile plastic orstainless steel containers for transfer to a sterile fill area forfilling into final containers, or the bulk serial was sampled and drawnoff into sterile plastic carboys. If the serial was drawn off intocarboys, it was stored at 4-8° C. If two or more carboys of bulk vaccinewere to be filled at a single time, the product was pooled into asterile stainless steel vessel when required for filling. Each dose ofvaccine was formulated to contain sufficient inactivated virus harvestfluids to provide at least 64 hemagglutination units (HAU) ofNewmarket/77 and 128 HAU of Newmarket/2/93 and 128 HAU of Kentucky/95.

EXAMPLE 3 Method of Monitoring Inactivation of Viruses

Each lot of EHV-1, EHV-4, EIV Newmarket/77, and EIV Kentucky/95 wastested for inactivation by passage in EVero cells. Each lot of EIVNewmarket/2/93 was tested for inactivation by passage in MDCK cells. Onehundred and fifty (150) cm² of EVero cell culture monolayer wereinoculated with 1.0 ml of inactivated EHV or EIV fluids and maintainedat 36° C.±2° C. for 14 days with at least two passages. At the end ofthe maintenance period, the cell monolayers were examined for CPEtypical of EHV or EIV. For EHV controls, a culture flask of EVero cellswas inoculated with reference EHV-1 or EHV-4 (positive control) to givea target multiplicity (MOI) of 0.001. For EIV controls, a culture flaskof EVero cells was inoculated with reference EIV Newmarket/77 or EIVKentucky/95 virus (positive control) to give a target MOI of 0.01. Forthe EIV Newmarket/2/93 tests, a culture flask of MDCK cells wasinoculated with reference EIV Newmarket/2/93 to give a target MOI of0.01. As a negative control, a uninoculated flask with EVero cells orMDCK cells was incubated under the same conditions as the testculture(s). After incubation and passage, the absence of virus-infectedcells in the BEI treated viral fluids constituted a satisfactoryinactivation test. The control cells inoculated with the reference virusshould show CPE typical of EHV or EIV. The uninoculated flask shouldshow no evidence of EHV or EIV CPE. After a satisfactory inactivationtest, residual BEI was neutralized by the addition of a cold sodiumthiosulfate solution and the inactivated fluids were stored at 4° C.±2°C. or below prior to bulk blending of the final product.

EXAMPLE 4 EHV Potency Release Assay

Coating of 96-well plates with EHV-1 mAb or EHV-4 mAb

96-well plates used for testing the potency of EHV-1 fractions werecoated with the EHV-1 monoclonal antibody. 96-well plates used fortesting the potency of EHV-4 containing samples were coated with theEHV-4 monoclonal antibody. The EHV-1 monoclonal antibody (“EHV-1mAb”),16H9, IgG fraction, was diluted 1:10,000 in coating buffer. The EHV-4monoclonal antibody (“EHV-4 mAb”), 20F3, IgG fraction, was diluted1:10,000 in coating buffer. Aliquots (100 μl) of the mAb solutions wereadded to all wells of the NUNC MAXISORP plates except the wells incolumn 1 and the plates were sealed with plate sealing covers. Themultiwell plates were then incubated for 1 hour at 37° C. and overnightat 4° C.

Quantification of EHV-1 or EHV-4 Antigen in Test Samples.

EHV-1 or EHV-4 antigens in test samples were quantified using microtiterplates coated with the respective mAb. Prior to testing in the ELISA,one mL aliquots of test samples (e.g., adjuvanted bulk vaccine or finalcontainer vaccine) in conical microfuge tubes were frozen at −40° C. orbelow for a minimum of 18 hours. On the day the ELISA was performed, thefrozen sample(s) of test vaccine in microfuge tubes and the frozen vialof the vaccine reference were quickly thawed in a 37° C. water bath andvortexed to resuspend settled material. An aliquot of 2.5 μl Triton®X-100 was added to the microfuge tube of vaccine reference and eachmicrofuge tube of test vaccine. The microfuge tubes were vortexed andincubated at room temperature for one hour. Tubes were vortexed every 15minutes. A 100 μl aliquot of the EHV-1 external reference control (orEHV-4 external reference control) was added to 900 μl of antigendiluent. An aliquot (2.5 μl) Triton® X-100 was added to the dilutedEHV-1 external reference. The external reference fluids were incubatedat room temperature for 1 hour and vortexed every 15 minutes.

During this one hour incubation, EHV mAb-coated plates were washed threetimes with PBS-Tween®. Remaining reactive sites in the wells wereblocked by post-coating all the wells with 200 μl/well of blockingbuffer. Plates were incubated with blocking buffer at 37° C. for aminimum of 30 minutes. After the one hour incubation period, two-foldserial dilutions of the vaccine reference and test vaccine were preparedby transfer of 500 μl of vaccine reference and test sample to a testtube containing 500 μl antigen diluent.

The post-coated plates were washed three times with PBS-Tween® solution.Aliquots (50 μl) from the undiluted through 1:32 dilutions of thereference and test vaccine were added to duplicate wells of EHV-1 mAband EHV-4 mAb coated plates as shown in Table IV-1. Aliquots of 50 μl ofthe appropriate external reference control were added to wells of eachplate as shown in Table IV-1. Plates were incubated at 37° C. for 1hour. During this one hour incubation, horse anti-EHV-1 serum (or horseanti-EHV-4 serum) was diluted 1:1,000 in antibody diluent and incubatedfor one hour at room temperature.

After the one hour incubation, the multiwell plates were washed threetimes with PBS-Tween® solution. Aliquots (50 82 l) of the diluted horseanti-EHV-1 serum (or diluted horse anti-EHV-4 serum) were added to allwells of the appropriate plate except wells in column 1. Plates werethen incubated at 37° C. for 1 hour. During this one hour incubation,sheep anti-horse IgG●HRP conjugate was diluted 1:2,500 in antibodydiluent and incubated for one hour at room temperature.

After the one hour incubation, plates were again washed three times withPBS-Tween® solution. Aliquots (50 μl) of the sheep anti-horse horseIgG●HRP conjugate were added to all wells of the plate except those incolumn 1. Plates were incubated at 37° C. for 1 hour. After the one hourincubation, plates were again washed three times with PBS-Tween®solution.

TMB substrate is prepared according to manufacturers instructions.Aliquots (100 μl) of TMB substrate solution were added with amultichannel pipetter to all wells in row A and then in order to rows Bthrough H of the plate. The multiwell plates were then incubated at roomtemperature. The optical densities of the wells were determined byreading the plate on a microplate reader at a wavelength of 650 nm. Thecontrol wells in column 1 served as blanks. Plates used forquantification of EHV-1 antigen were read 35±10 minutes after theaddition of TMB substrate. Optical densities in wells containing theEHV-1 external reference should be between 0.500 and 1.200. Plates usedfor quantification of EHV-4 antigen were read 45±10 minutes after theaddition of TMB substrate. Optical densities in wells containing theEHV-4 external reference should be between 0.500 and 1.200. Relativepotency values (“R PV”) of test vaccine samples were determined from theoptical density readings by normalizing values against the EHV-1external reference control (or EHV-4 external reference control).

Criteria for a Valid Test

The optical density in wells containing the EHV-1 external reference (orEHV-4 external reference) should be between 0.500 and 1.200 30±5 minutesafter addition of TMB substrate. The optical density in the negativecontrol wells should be no more than 0.250. If either of these validitycriteria were not satisfied, the assay should be considered a NO TESTand the assay may be repeated without bias. The relative potency resultsof an assay were considered satisfactory if RPV (for inactivated EHV-1and/or inactivated EHV-4) of a unit dose of the test sample was greaterthan or equal to 1.0.

Reagents

EHV-1 specific monoclonal antibody, IgG fraction. Hybridoma 16H9 wasobtained from Dr. George Allen, Gluck Equine Research Center, Universityof Kentucky, Lexington, Ky. The IgG fraction from mouse ascites wascommercially prepared. The purified antibody was identified as EHV-1 mAb16H9-IgG, Lot 001, 11-11-98, Exp. 11-11-03. The IgG antibody fractionwas stored at 4-8° C.

EHV-4 specific monoclonal antibody, IgG fraction. Hybridoma 20F3 wasobtained from Dr. George Allen, Gluck Equine Research Center, Universityof Kentucky, Lexington, Ky. The IgG fraction from mouse ascites wascommercially prepared. The purified antibody was identified as EHV-4 mAb20F3-IgG, Lot 001/11-11-98, Exp. 11-11-03. The IgG antibody fraction wasstored at 4-8° C.

Horse anti-EHV-1 polyclonal serum. A pool of serum was prepared fromblood collected from the non-vaccinated control horses in the EHVImmunogenicity Study, 623-510-98E-015, at 21 days post challenge withvirulent EHV-1. The antibody was identified as Horse anti-EHV-1, Lot001/030199, Exp. 030104. The serum was stored frozen at −40° C. orbelow.

Horse anti-EHV-4 polyclonal serum. A pool of serum was prepared fromblood collected from the non-vaccinated control horses in the EHVImmunogenicity Study, 623-510-98E-015, at 21 days post challenge withvirulent EHV-4. The antibody was identified as Horse anti-EHV-4, Lot001/030399, Exp. 030304. The serum was stored frozen at −40° C. orbelow.

Sheep anti-horse IgG●HRP conjugate, 1 mg/ml, Bethyl Laboratories,Inc.Catalog No. A70-121P. The antibody conjugate was stored at 4-8° C.

EHV-1 external reference fluids. EHV-1 fluids were produced according tothe procedure described in Example 1. Vials of EHV-1 external referencefluids were identified as EHV-1 Ext. Ref. Flds., Lot 001/040599. Exp.040504. Virus fluids were stored at −40° C. or below.

EHV-4 external reference fluids. EHV-4 fluids were produced according tothe procedure described in Example 6. Vials of EHV-4 external referencefluids were identified as EHV-4 Ext. Ref. Flds., Lot 001/040699. Exp.040604. Virus fluids were stored at −40° C. or below.

EHV-1/EHV-4 reference vaccine. The EHV-1/EHV-4 reference vaccine is thesame vaccine used in the Immunogenicity study described in Example 8(623-0510-98E-015). The vaccine was identified as EHV Imm. Vac,623-510-98E-015, lot# 001, 11-6-98. Exp. 110603. The vaccine referencewas stored at −40° C. or below.

The following commercially available reagents were used in theexperiment:

Substrate system. TMB (two component), Kirkegaard and Perry, catalog no.50-76-00.

Triton® X-100. Sigma, catalog no. 1043.

Bovine calf serum. Hyclone Laboratories, Inc., catalog no. A-2151-L.

The following standard solutions were prepared for use in theexperiment:

Coating Buffer (Prepared fresh for each test and adjusted to pH 9.6)g/liter deionized H₂O a. Na₂CO₃•anhydrous 159 b. NaHCO₃ 2.93

PBS (adjusted to pH 7.3) g/liter deionized H₂O a. Na₂HPO₄•anhydrous 118b. NaH₂PO₄•anhydrous 0.23 c. NaCl 50

PBS-Tween® Solution g/liter deionized H₂O a. Na₂HPO₄•anhydrous 18 b.NaH₂PO₄•anhydrous 20 c. NaCl 50.8 d. Tween ® 20 0.50 ml

Blocking buffer (Prepared fresh on day of test) a. PBS 75 ml b. Bovinecalf serum 25 ml

Antibody diluent (Aame as blocking buffer; prepared fresh on day oftest).

Antigen diluent (Prepared fresh on day of test) a. PBS   50 ml b.Triton ® X-100 0.05 ml

EXAMPLE 5 Inoculation of Horses with Inactivated EHV-1 and SubsequentChallenge with Virulent EHV-4

The purpose of the study was to demonstrate immunogenicity of aninactivated EHV-1 KyA virus for cross protection of vaccinated horseschallenged with virulent EHV-4. The vaccine used in the study includedwith inactivated EHV-1 KyA virus adjuvanted with Carbopol® 971. Horseswere vaccinated by two different vaccination regimens. One vaccinationregimen was three intramuscular vaccinations and the other vaccinationregimen was two intramuscular vaccinations and one intranasaladministration. Horses were vaccinated at two to four week intervals.Non-vaccinate horses served as controls. At three weeks after the lastvaccination, vaccinated and non-vaccinated control horses werechallenged with virulent EHV-4.

Severe respiratory disease was observed in non-vaccinated control horsespost challenge with EHV-4. Horses vaccinated by either the intramuscularor intramuscular/intranasal regimens with the EHV-1 KyA vaccine showed asignificant reduction in clinical signs of respiratory disease caused byEHV-4 compared to non-vaccinated and challenged control horses. Therewas a significant reduction in virus shedding between horses vaccinatedby either vaccination regimen versus non-vaccinated control horses. Theresults of the study demonstrated the immunogencity of the inactivatedEHV-1 KyA fraction of the vaccine against respiratory disease caused byvirulent EHV-4. Moreover, the study demonstrated that the inactivatedEHV-1 KyA fraction was immunogenic when administered by both theparenteral and parenteral/intranasal routes.

Healthy male and female horses that ranged in age from four to ninemonths were obtained from selected sources. Horses were identified byhalter tag numbers and microchip numbers. All horses were in good healthat the start of the study. At the time of the first vaccination, horseshad virus neutralization (VN) titers of ≦2 to EHV-1 and EHV-4. Horseswere randomized into groups by drawing the identification numbers of thehorses from a bag. During the vaccination and challenge periods, horseswere maintained together in open pens and were fed free choice dairyquality alfalfa hay, Sweet 14 dietary supplement, equine Bio-mineral,and water ad libum.

Horses were randomized into groups by drawing horse identificationnumbers from a bag. Horses were observed for general health and anyabnormal behavior during the vaccination period. No abnormal behavior oradverse health conditions were observed in any of the horse postvaccination and no adverse injections site reactions were observed inany horse post vaccination. No clinical signs of respiratory diseasewere observed in any of the horses during the vaccination period.

EHV-1 fluids for use in the vaccine were produced according to theprocedure described in Example 1. All virus fluids were at the fifthpassage from master seed virus and were produced on cells at the 20^(th)passage from master cell stock. The vaccine was formulated to containEHV-1 and EIV Newmarket/77, A1 subgroup, EIV Kentucky 95, A2 subgroupand Newmarket 2/93, A2 subgroup, per two ml dose (produced according tothe procedure described in Example 2). The vaccine was labeled as EHV-1Imm Vac, 623-510-99E-116, lot # 001, Dec. 19,1999.

The immunogenic composition containing inactivated EHV-1 wasadministered to horses by two vaccination regimens. One vaccinationregimen was three intramuscular vaccinations at three week intervals.The other regimen was two intramuscular inoculations at two-four weeksapart followed by a third inoculation by the intranasal route two tofour weeks later. Each vaccination regimen group contained 11 or morehorses. A group of 18 horses served as non-vaccinated controls. At threeweeks after the last vaccination, horses were challenged with virulentEHV-4. Horses were monitored for clinical signs of respiratory diseasecaused by EHV-4 and the severity of the disease was recorded by ascoring system described below in Table V-1. Blood and nasal samples forserological evaluation and nasal swabs for isolation of virus were takenbefore and at selected times post vaccination. TABLE V-1 Clinical SignsScoring System Clinical sign Score given for each day exhibiting signNasal discharge serous, slight amount 1 serous copious amount 2mucopurulent, slight amount 3 mucopurulent, copious amount 4 Pyrexia102.5-103.9° F. 1 104.0-104.9° F. 2 ≧>105.0° F. 3 Other SymptomsConjunctivitis 1 Coughing 2 Dyspnea 3 Depression 4 Antibiotic treatmentrequired 5 for secondary bacterial infection

Five replicate titrations of EHV-4 challenge virus, strain 405 wereconducted on titrations. Two ml was administered for challenge to give4.9 TCID₅₀ Log₁₀/dose. This dose of virulent virus was sufficient tocause severe clinical respiratory disease in non-vaccinated controlhorses.

EHV-4 405 strain was obtained from the American Type Culture Collectionand was propagated on Vero cells. This virus was isolated from a horsewith rhinopneumonitis and was characterized by Dr. M. Studdert, a wellrecognized scientist in EHV and diseases caused by EHV. The virus wassubmitted to ATCC as a relevant and representative example of EHV-4 andhas previously been recommended by NVSL as an EHV-4 challenge strain.

Frozen stocks of EHV-4 to be used for challenge were thawed and dilutedto contain a target concentration of 5.0 TCID₅₀ log₁₀/ml and frozen at−70° C. A sample of challenge virus was thawed and the titer determined.At the time of challenge, the virus fluids were thawed and two ml ofEHV-4 was withdrawn with a three ml syringe and 21 gauge needle. Theneedle was removed and the challenge virus was administered intranasallyto appropriate groups of vaccinated and non-vaccinated horses.

Post challenge, horses were monitored for clinical signs of respiratorydisease cause by EHV-4 that included pyrexia, nasal discharge,conjunctivitis, coughing, dyspnea, depression, and other such asantibiotic treatment required for secondary bacterial infection. Horseswere observed daily for clinical disease and the severity of disease wasrecorded.

Nasal swabs for isolation of EHV were taken on selected days post viruschallenge and stored at −70° C. Frozen samples were thawed and the swabremoved from the transport medium. The sample was processed bycentrifugation at 2500 ×g for 20 minutes at 19-22° C. An aliquot of 0.1ml of the processed sample was added to a well of a 48-well tissueculture plate containing 24 hour monolayers of Vero cells. Cultureplates were incubated at 37° C. in a CO₂ incubator for seven days. Wellswere observed on a regular basis for the presence of cytopathic effect(CPE) typical of EHV. Wells that exhibited cytotoxicity were subculturedafter the seven day incubation period by transfer of 0.2 ml from thewell to a well of a 48-well tissue culture plate containing a 24 hourmonolayer of Vero cells. Culture plates were incubated at 37° C. in aCO₂ incubator for seven days and observed for CPE. The titer of EHV inthe processed nasal samples that were positive for CPE was determined bystandard titration methods on cells in 96-well tissue culture plates.

Serum VN antibody titers in horses before and after vaccination andafter challenge with virulent virus were determined. When blood sampleswere taken to screen horses for use in the study, all horses had VNantibody titers of ≦2 for EHV-1 and EHV-4, except horse number 13, thathad a VN titer of 8 to EHV-1 and EHV-4. At the time of the firstvaccination, the VN titer to EHV-1 and EHV-4 had declined to 2 and 4respectively. One additional horse in the intramuscular group had a VNtiter that had increased from 2 to 8 and two horses in theparenteral/intranasal group had VN titers that had increased from 2 to16. None of the horses showed any evidence of a respiratory infection.Post first vaccination, the increase in VN titer to EHV-1 and EHV-4 wasvariable but the VN titer in all horses increased relative to thepre-vaccination titer. After the second and third vaccinations, the VNtiter remained essentially the same as after post first vaccination. TheVN titer was greater to EHV-1 than to EHV-4, although the VN titer toEHV-1 was greater than two-fold but less than four-fold greater than theEHV-4 VN titer. The VN titers to EHV-1 and EHV-4 were greater in theparenteral vaccination group than the parenteral/intranasal group but byless than a two-fold difference. At 21 days post challenge with virulentEHV-4, VN antibody titers to both EHV-1 and EHV-4 had increased slightlyor remained the same in the vaccinated horses. However, innon-vaccinated control horses, VN antibody titers increased 32 to 128fold to both EHV-1 and EHV-4 in all but one non-vaccinated controlhorses. VN antibody titers to EHV-1 were similar to VN antibody titersto EHV-4 in vaccinated and control horses post EHV4 challenge.

VN antibody titers in nasal samples collected from horses before andafter vaccination and after challenge with virulent virus weredetermined with the same VN assay used to determine antibody titers inserum. Similar to VN activity in serum post vaccination, VN activity innasal secretions increased after vaccination but to a lesser extent.Post challenge with EHV-4, VN antibody titer increased but only slightlyand only in a few horses. VN antibody titer data are not presented inthe report.

Horses were challenged intranasally with virulent EHV-4. Post challenge,temperature of horses were taken daily and clinical signs of respiratorydisease that included nasal discharge, included conjunctivitis,coughing, dyspnea, and depression were recorded.

Pyrexia in horses post EHV-4 challenge was monitored. Pyrexia wassporadic and of short duration in both vaccinate groups and in thecontrol group. In the majority of horses that exhibited pyrexia, theelevated temperature was observed for only one or two days. Onevaccinate horse in the parenteral/intranasal group exhibited pyrexia forfour consecutive days. There was no evidence of significant differencesin the distribution of pyrexia scores among vaccinate and control groupsfor days 5 through 8, 10 or 13 post challenge. Statistical analysis wasnot performed on days 1 through 4, 9, 11, 12, and 14 through 21 becauseless than two animals had nonzero pyrexia scores. When temperatures wereanalyzed separately by day using analysis of variance there was evidenceof significant reduction of temperature among vaccinate and controlgroups but only for a few individual days.

Observations of nasal discharge in horses were also made. Nasaldischarge was assessed as normal, serous slight amount, serous copiousamount, mucopurulent slight amount, and mucopurulent copious amount andscored as 0, 1, 2, 3, and 4 respectively. Three horses in the parenteralvaccinate group exhibited mucopurulent nasal discharge for only one day.All other discharge scores in vaccinate groups were slight of amount ofserous discharge that was primarily limited to one o two days. Incontrast, non-vaccinated control horses exhibited mucopurulent andserous discharge for multiple consecutive days. There was evidence ofsignificant differences among both vaccinate groups and controls(≦0.0479) for days 3 through 9 and 11 through 15 post challenge. Therewas evidence of significant differences in the distribution of nasaldischarge scores between the parenteral vaccination group and controlgroup (p≦0.0398) for days 4,5,6,8,9, and 11 post challenge and betweenthe parenteral/intranasal vaccination and control groups (p≦0.0259) days3 through 8 and 11 post challenge.

Post challenge observations of clinical signs of conjuctivitis,coughing, dyspnea, and depression are presented in Table 4.Conjuctivitis was the primary sign observed in vaccinates and wasrecorded for two to three days. One vaccinate exhibited dyspnea for oneday and one vaccinated exhibited coughing for three consecutive days.All other observations of coughing were for one day only. One horse eachin the parenteral and parenteral/intranasal vaccinates groups, 8% and9%, respectively exhibited two signs of clinical disease for only oneday. Depression was not observed in any vaccinate. In distinction tovaccinates, 80% of the non-vaccinated control horses exhibited severeclinical signs of disease of coughing and depression for three or moreconsecutive days post challenge. When clinical signs were analyzed as0=absent and 1=present, there was evidence of a significant differencein number of animals exhibiting conjuctivitis between the parenteral(p≦0.0313) and parenteral/intranasal (p≦0.0391) vaccination groups andthe non-vaccinated control group for days 3 and 6 through 13 postchallenge. There was evidence of a significant difference in reductionof animals exhibiting depression (p≦0.0156) on day 6 post challenge forboth the parenteral and parenteral/intranasal vaccinate groups comparedto non-vaccinated controls. There was no evidence of a significantdifference between vaccinates and controls for dyspnea.

When number of clinical signs exhibited each day were tallied, there wasevidence of significant difference in the distribution of number ofclinical signs between parenteral (p≦0.0242) and parenteral/intranasal(p≦0.0259) vaccinate groups and non-vaccinated control horses on days 6through 9 and day 11 post challenge. Clinical sign scores ofconjuctivitis, coughing, dyspnea, and depression were scored 1,2,3, and4, respectively as described in Table V-1. A composite clinical scoreswas calculated by a sum of the nasal discharge and clinical sign scores.Temperatures were not included in the calculation of composite score.The composite clinical score was calculated for each horse for each daypost challenge. The total composite clinical score is the sum of theclinical scores for all post challenge days. There was evidence ofsignificant differences in composite clinical score between theparenteral and non-vaccinated control group (p≦0.0331) on days 3 through9 and 11 through 15 post challenge and significant differences for totalcomposite score (p≦0.0001). There was evidence of significantdifferences in composite clinical score between theparenteral/intranasal and non-vaccinated control group (p≦0.0241) ondays 3 through 9 and 11 through 15 post challenge and significantdifferences for total composite score (p≦0.0001).

Virus shedding was monitored in horses after challenge with virulentEHV-4. Nasal samples were collected from horses on days 1 through 7 andevery other day until 18 days post challenge. EHV-4 challenge virus wasrecovered from nasal samples from all but one non-vaccinated controlhorse. Virus was detected from the majority of non-vaccinated controlhorses at the 10⁻² dilution. Days 3 through 5 were the major days whenvirus shedding was detected. Virus was detected from some non-vaccinatedcontrol horses on day 6 post challenge but at low levels. Challengevirus was not recovered from vaccinated horses post challenge.

CONCLUSION

The vaccine containing inactivated EHV-1 KyA virus generated a systemichumoral immune response when administered by both the parenteral andparenteral/intranasal routes. Vaccination of horses with the EHV-1containing vaccine generated high levels of VN antibody to EHV-1 and toEHV-4. Antibody titers to both EHV-1 and EHV-4 were detected in nasalsamples from vaccinated animals but at a relatively low titer. Thus, theinactivated EHV-1 KyA containing vaccine was capable of immunizinghorses against EHV-1 and was capable of cross-immunizing horses againstEHV-4. No abnormal response to vaccination or reaction site injectionswere observed in an of the horses post vaccination.

Severe respiratory disease that consisted of prolonged episodes ofserous and mucopurulent nasal discharge, conjuctivitis, coughing anddepression was observed in non-vaccinated control horses challenged withvirulent EHV-4. In contrast, the number of clinical signs of EHV-4respiratory disease, the severity of the clinical signs and the numberof vaccinates exhibiting clinical signs were significantly reduced invaccinated horses. Horses vaccinated by either the parenteral orparenteral/intranasal routes showed a significant reduction in clinicalsigns of respiratory disease due to EHV-4 infection. Reduction inclinical disease was supported by data establishing that both parenteraland parenteral/intranasal vaccinated horses did not shed virus postEHV-4 challenge. Nasal samples collected from non-vaccinated controlhorses contained high levels of virus on multiple days post EHV-4challenge.

The inactivated EHV-1 KyA antigen contained in the vaccine wasimmunogenic for cross protection of horses against respiratory diseasecaused by EHV-4 when administered by the parenteral andparenteral/intranasal routes. In summary, the results of this studydemonstrated that a vaccine containing inactivated EHV-1 KyA virusgenerated VN antibody not only to EHV-1 but cross neutralizationantibody to EHV-4. The inactivated EHV-1 KyA containing vaccine wascapable of cross protection of horses against respiratory disease causedby virulent EHV-4 when administered using either a parenteral orparenteral/intranasal regime.

EXAMPLE 6 Inoculation of Horses with Inactivated EHV-1 and SubsequentChallenge with Virulent EHV-1

The objective of the study described in this example was to demonstratethe immunogencity of the EHV-1 fraction, when administered by either theintramuscular or intramuscular/intranasal routes, for cross protectionof horses against disease caused by virulent EHV-1. An additionalobjective was to demonstrate non-interference of the EIV fractionspresent on the immunogenicity of EHV-1.

The purpose of the study was to demonstrate immunogenicity of the EHV-1fraction of the rhinopneumonitis vaccine, killed virus for protection ofvaccinated horses challenged with virulent and EHV-1. The vaccine usedin the study was formulated with inactivated EHV-1 and adjuvanted withCarbopol® 971. Horses were vaccinated by two different vaccinationregimens. One vaccination regimen was three intramuscular vaccinationsand the other vaccination regimen was two intramuscular vaccinations andone intranasal administration. Horses were vaccinated at two to fourweek intervals. Non-vaccinate horses served as controls. At six weeksafter the last vaccination, vaccinated and non-vaccinated control horseswere challenged with virulent EHV-1. Clinical signs of severerespiratory disease that included mucopurulent nasal discharge,coughing, dyspnea, and depression were observed in non-vaccinatedcontrol horses post challenge with EHV-1.

Horses

Healthy male and female horses of different breeds, three to four monthsof age were obtained from several sources. Horses were identified byhalter tag numbers and microchip numbers. All horses were in good healthat the start of the study with no known previous incidence ofrespiratory disease caused by EHV. Horses were randomized into groups bydrawing the identification numbers of the horses from a bag. During thevaccination and challenge periods, horses were maintained together inopen pens and were fed free choice dairy quality alfalfa hay, Sweet 14dietary supplement, equine Bio-mineral, and water ad libum.

Horses were randomized into groups by drawing horse identificationnumbers from a bag. Horses were observed for general health and anyabnormal behavior during the vaccination period. All horses were in goodhealth at the start of the study. No abnormal behavior or adverse healthconditions were observed in any of the horse post vaccination and noadverse injections site reactions were observed in any horse postvaccination. No clinical signs of respiratory disease caused by EHV wereobserved in any of the horses during the vaccination period.

At approximately three weeks before the intended date of challenge ofhorses with virulent EHV-1, an outbreak of Strangles occurred in thehorses. Samples taken from swollen lymph nodes were submitted to MontanaState University diagnostic laboratory and Streptococcus equi wasisolated from the samples. Penicillin was administered to the horses andhorses were vaccinated with a live attenuated S. equi vaccine, Pinnacle.Horses were allowed to recover from Strangles for three weeks beforechallenge with virulent EHV-1. One horse, a non-vaccinated controlhorse, was removed from the study on the day of challenge with EHV-1.Slight depression and dyspnea were observed and inspiratory squeaks wereheard upon examination. Another horse, in the intramuscular vaccinategroup, died Sep. 21, 2000, five weeks prior to EHV-1 challenge. Thecause of death was pneumonia. A third horse, in the intramuscularvaccinate group, died on Oct. 28, 2000, one day post challenge. Thecause of death was a ruptured mesentery abscess and subsequent toxemia.S. equi was isolated from the abscess. All horses challenge with EHV-1were healthy and showed no evidence of S. equi infection. All data fromthe horses removed from the study were not included in the report.

Vaccine

EHV-1 fluids for use in the vaccine were produced according to theprocedure described in Examples 1 and 2. All virus fluids were at thefifth passage from master seed virus and were produced on cells at the20^(th) passage from master cell stock. The vaccine was formulated tocontain EHV-1 and EIV Newmarket/77, A1 subgroup, EIV Kentucky 95, A2subgroup and Newmarket 2/93, A2 subgroup, per two ml dose. The vaccinewas labeled as EHV-1 Imm Vac, Lot #001, 6129-0510-00E-045, 03 Aug. 2000.

Study Design

The vaccine was administered to horses by two vaccination regimens. Onevaccination regimen was three intramuscular vaccinations at two to fourweek intervals. The other regimen was two intramuscular vaccinations attwo-four weeks apart and the third vaccination by the intranasal routetwo to four weeks later. Each vaccination regimen group contained 19-20horses. A group of 20 horses served as non-vaccinated controls. At sixweeks after the last vaccination, horses were challenged with virulentEHV-1. Horses were monitored for clinical signs of respiratory diseasecaused by EHV-1 and the severity of the disease was recorded. Blood andnasal samples for serological evaluation and nasal swabs for isolationof virus were taken before and at selected times post vaccination.Collection of blood, nasal samples, and nasal swabs are described in theanimal study protocol.

Serum VN Antibody Titers to EHV-1 and EHV-4

Serum VN antibody titers in horses before and after vaccination andchallenge with virulent EHV-1 were determined. All horses in both theintramuscular and intramuscular/intranasal vaccinate groups had VNantibody titers of ≦2 or 4 to EHV-1 at the time of the firstvaccination. One horse in the intramuscular vaccinate group and fivehorses in the intranasal vaccinate group had VN antibody titers to EHV-4of 8 or 16 at the time of the first vaccination. VN antibody titers inthe non-vaccinated control horses were ≦2 to 16 with the majority at ≦2or 4 at the time of the first vaccination. None of the horses showed anyevidence of a respiratory infection and there was no known previousexposure to EHV. After one vaccination, there was little to no increasein VN titer to EHV-1 and EHV-4. Antibody titers in the non-vaccinatedcontrol horses remained unchanged and, in fact, antibody titers in somecontrols decreased slightly from pre vaccination titers. After thesecond vaccination, VN antibody titers to EHV-1 increased in theintramuscular and intramuscular/intranasal vaccinate groups. Antibodytiters to EHV-4 did not increase in either of the vaccinate groups.Antibody titers in non-vaccinated controls remained unchanged orcontinued to decline in the non-vaccinated control group. Post thirdvaccination, the VN titers to EHV-1 continued to increase. Antibodytiters to EHV-4 also increased after the third vaccination. Thegeometric mean VN antibody titers to EHV-1 and EHV-4 in theintramuscular vaccinate group were 86 and 19, respectively and were 69and 20 for EHV-1 and EHV-4, respectively in the intramuscular/intranasalgroup. With the exception of three non-vaccinated control horses,antibody titers to EHV-1 and EHV-4 remained unchanged or declined by theend of the third vaccination period. At 21 days post challenge withvirulent EHV-1, VN antibody titers to both EHV-1 and EHV-4 increaseddramatically in some vaccinates and increased only slightly or remainedthe same in other vaccinates. However, in non-vaccinated control horses,VN antibody titers increased over 100 fold to both EHV-1 and EHV-4 inall but four non-vaccinated control horses. In general, VN antibodytiters were greater to EHV-1 than to EHV-4.

Titration of EHV-1 Challenge Virus

Five replicate titrations of EHV-1 challenge virus, strain KyD wereconducted on Vero cells. Results of the five replicate titrations were4.4, 4.4, 4.4, 4.4, and 4.5 TCID₅₀Log₁₀/ml. The mean titer was 4.4TCID₅₀ Log₁₀/ml. Two ml was administered to each horse for challenge togive 4.7 TCID₅₀ Log₁₀/2 ml dose. The target dose of virus to beadministered to horses for challenge was ≧4.0 TCID₅₀ Log₁₀/2 ml dose.

Challenge of Horses with Virulent EHV-1

EHV-1 KyD strain was obtained from the American Type Culture Collectionand was propagated on Vero cells. Stocks of the EHV-1 to be used forchallenge were to contain a target concentration of ≧4.0 TCID50log₁₀/ml. Stocks were frozen at −70° C. A sample of challenge virus wasthawed and the titer determined. At the time of challenge, the virusfluids were thawed and two ml of the EHV-1 was withdrawn with a three mlsyringe and 21 gauge needle. The needle was removed and the challengevirus was administered intranasally to appropriate groups of vaccinatedand non-vaccinated horses. Vaccinates and non-vaccinated control horseswere housed together in open pens during the 21 day post challengeperiod.

Post challenge, horses were monitored for pyrexia and clinical signs ofrespiratory disease cause by EHV-1 that included, nasal discharge,conjunctivitis, coughing, dyspnea, depression, and other such asantibiotic treatment required for secondary bacterial infection. Horseswere observed daily for clinical disease and the severity of disease wasrecorded.

Pyrexia in horses post EHV-1 challenge was monitored. Pyrexia wassporadic and was spread out over the duration of the challenge period.There were horses in the non-vaccinated control group as well as bothvaccinate groups that had elevated temperatures for two and threeconsecutive days but there did not seem to be a correlation of pyrexiawith clinical signs of respiratory disease. There was no evidence ofsignificant differences in the distribution of pyrexia scores betweeneach of the vaccinate groups and the non-vaccinated control group.

Observations of nasal discharge in horses were made. Nasal discharge wasrecorded as normal, serous slight amount, serous copious amount,mucopurulent slight amount, and mucopurulent copious amount and scoredas 0, 1, 2, 3, and 4 respectively, according to the animal protocol.Serous nasal discharge was observed post challenge in all horses in theintramuscular vaccinate group with about an equal number of horsesexhibiting slight and copious amounts of serous discharge. Horses 405and 423 had two consecutive days of mucopurulent discharge and horses420 and 469 had one day of mucopurulent discharge. Non-vaccinatedcontrol horses exhibited primarily mucopurulent discharge for multipleconsecutive days. When nasal discharge scores are analyzed as per thescoring system in the animal protocol, there is evidence of asignificant difference in the distribution of nasal discharge scoresbetween the intramuscular group and non-vaccinated controls group ondays 5-7 and 10 post challenge (p≦0.0018). Similar results of serousnasal discharge were observed in the intramuscular/intranasal group postchallenge with mucopurulent discharge recorded for two vaccinates forone or two days. There is evidence of a significant difference in thedistribution of nasal discharge scores between theintramuscular/intranasal group and non-vaccinated controls group on days4-7, 10 and 19 post challenge (p≦0.0463).

When nasal discharge scores were analyzed according to a system ofnormal=0, serous=1 and mucopurulent=2, there was evidence of asignificant difference between the intramuscular group andnon-vaccinated controls group on days 5-7, 10 and 17 post challenge(p≦0.0463). There was also evidence of a significant difference in thedistribution of nasal discharge scores between theintramuscular/intranasal group and non-vaccinated controls group on days4-7 and 10 post challenge (p≦0.0092).

Post challenge observations of clinical signs of conjuctivitis,coughing, dyspnea, and depression were also made. Days 3 through 11 postchallenge, were days when the most severe signs of disease were observedin horses. Clinical signs of disease occurred most frequently on days 3through 11 post challenge. There also seemed to be a secondary phase ofclinical signs that were observed in vaccinates and controls. However,this was observed in vaccinates for only single days and in controls formultiple days. Coughing and conjuctivitis were the primary signs ofclinical disease observed in vaccinates in the intramuscular regimengroup. Conjuctivitis and coughing were observed in the vaccinates for nomore than three consecutive days. There is evidence of a significantreduction in proportion of animals exhibiting conjuctivitis in theintramuscular group compared to non-vaccinated controls on days 6 and 7post challenge (p≦0.0197) and there is evidence of a significantreduction in proportion of animals exhibiting coughing in theintramuscular group compared to non-vaccinated controls on days 4through 8 post challenge (p≦0.0463). Conjuctivitis and coughing were themost common clinical signs of disease observed in theintramuscular/intranasal vaccinate group. There is evidence of asignificant reduction in proportion of animals exhibiting conjuctivitisin the intramuscular/intranasal group compared to non-vaccinatedcontrols on days 6 and 7 post challenge (p≦0.0197) and there is evidenceof a significant reduction in proportion of animals exhibiting coughingin the intramuscular group compared to non-vaccinated controls on days 5and 6 post challenge (p≦0.0044). Depression was not observed in anyintramuscular vaccinate post challenge and was observed in only twointramuscular/intranasal vaccinates post challenge. Depression wasobserved in multiple non-vaccinated control horses for multiple days. Incontrast to the vaccinates, non-vaccinated control horses demonstratedmultiple signs of disease that in general persisted for four consecutivedays and for as long as seven or eight days. Dyspnea was not observed inany vaccinated horse but was observed in two control horses for multipledays. The same two non-vaccinated control horses required antibiotictreatment for secondary bacterial infection. Clinical disease scores, ascalculated per the animal protocol are presented in tables 7 and 8 forintramuscular vaccinates versus controls and intramuscular/intranasalvaccinates versus controls, respectively.

The number of clinical signs were tallied for each day and reported as0, 1, 2, or 3. When number of clinical signs exhibited each day weretallied, there is evidence of significant difference in the distributionof number of clinical signs between intramuscular group andnon-vaccinated control group for days 4 through 8 (p≦0.0206. There isevidence of significant difference in the distribution of number ofclinical signs between intramuscular/intranasal group and non-vaccinatedcontrol group for days 5 through 7 (p≦0.0159).

A composite clinical scores was calculated by a sum of the nasaldischarge and clinical sign scores. The Kruskal-Wallis test, amultigroup extension of the two-group Wilcoxon's test was used to testthe hypothesis of equality of scores among groups. Wilcoxon's test wasused to test the hypothesis of reduction in scores for each vaccinategroup compared to the control group (a one-sided test) and to test thehypothesis of equality of scores between vaccinate groups (a two-sidedtest). There is evidence of significant reduction in composite clinicalscores between intramuscular group and non-vaccinated control group fordays 4 through 7 and day 11 post challenge (p≦0.0372) and for totalcomposite clinical score (p≦0.0001). There is evidence of significantreduction in composite clinical scores between intramuscular/intranasalgroup and non-vaccinated control group for days 4 through 7 and day 11post challenge (p≦0.0408) and for total composite clinical score(p≦0.0002).

A modified composite clinical score, the sum of clinical observationscore and nasal discharge score but not pyrexia, was also calculated.There was evidence of significant reduction in modified compositeclinical scores between intramuscular group and non-vaccinated controlgroup for days 3 through 8 and days 10, 11 and 19 post challenge(p≦0.0383) and for total composite clinical score (p≦0.0001). There wasalso evidence of significant reduction in composite clinical scoresbetween intramuscular/intranasal group and non-vaccinated control groupfor days 3 through 7 and days 10, 11 and 19 post challenge (p≦0.0487)and for total composite clinical score (p≦0.0001).

Shedding of Virus from Horses Post Challenge

EHV-1 challenge virus was recovered from nasal samples from allnon-vaccinated control horses shed virus post challenge with EHV-1 andstored at −70° C. Frozen samples were thawed and the swab removed fromthe transport medium. The sample was processed by centrifugation at2500×g for 20 minutes at 19-22° C. An aliquot of 0.1 ml of the processedsample was added to a well of a 48-well tissue culture plate containing24-48 hour monolayers of Vero cells. Culture plates were incubated at37° C. in a CO₂ incubator for seven days. Wells were observed on aregular basis for the presence of cytopathic effect (CPE) typical ofEHV. Wells that exhibited cytotoxicity were subcultured after the sevenday incubation period by transfer of 0.2 ml from the well to a well of a48-well tissue culture plate containing a 24 hour monolayer of Verocells. Culture plates were incubated at 37° C. in a CO₂ incubator forseven days and observed for CPE. The titer of EHV in the processed nasalsamples that were positive for CPE was determined by standard titrationmethods on cells in 96-well tissue culture plates.

EHV-1 challenge virus was recovered from nasal samples from allnon-vaccinated control horses shed virus post challenge with EHV-1.Virus detected in the nasal swabs was identified as EHV-1 by theimmunofluorescence assay using EHV-1 specific monoclonal antibody. Viruswas detected from non-vaccinated control horses at the 10⁻² to 10⁻³dilutions. Days two through five were the major days when virus sheddingwas detected. Challenge virus was recovered post challenge from fivehorses in the intramuscular vaccinate group and from three horses in theintramuscular/intranasal vaccinate group. There is evidence of asignificant reduction in proportion of animals exhibiting the presenceof virus in intramuscular group compared to non-vaccinated control groupfor days 2 through 5 (p≦0.0001) and there is evidence of a significantreduction in proportion of animals exhibiting the presence of virus inintramuscular/intranasal group compared to non-vaccinated control groupfor days 2 through 5 (p≦0.0003).

Criteria for a Satisfactory Study

The following criteria must be met for a satisfactory EHV immunogenicitystudy:

-   -   Non-vaccinated control horses must remain seronegative to EHV-1        and EHV-4 during the vaccination period and/or not show any        clinical sign of disease as an indicator of exposure of test        horses to virulent field virus.    -   Post challenge with virulent EHV-1, there must be a        statistically significant reduction in clinical signs of disease        in vaccinated animals compared to clinical signs of disease in        non-vaccinated control animals.

CONCLUSIONS

In this study, groups of male and female foals of three to four monthsof age were vaccinated with three doses of vaccine administered byeither intramuscular or intramuscular/intranasal routes. Following theadministration of three doses of vaccine, all vaccinated animalsdeveloped VN antibody. Development of the systemic humoral response wassimilar in animals vaccinated by the intramuscular andintramuscular/intranasal vaccination regimens. Vaccination of horseswith the EHV-1 containing vaccine generated high levels of VN antibodyto EHV-1 and to EHV-4. Thus, the EHV-1 containing vaccine was capable ofimmunizing horses against EHV-1 and was capable of cross-immunizinghorses against EHV-4. No abnormal response to vaccination or reactionsite injections were observed in an of the horses post vaccination.

To evaluate the ability of the EHV-1 containing vaccine to protecthorses against respiratory disease caused by EHV-1, clinical signs ofrespiratory disease in vaccinated horses were compared to clinicaldisease in non-vaccinated control horses after challenge with a virulentstrain of EHV-1 strain KyD. At approximately six weeks post thirdvaccination with the EHV-1 containing vaccine, vaccinated andnon-vaccinated control horses were challenged intranasally with virulentEHV-1. Severe respiratory disease that consisted of prolonged episodesof serous and mucopurulent nasal discharge, conjuctivitis, coughing,depression, and dyspnea were observed in non-vaccinated control horseschallenged with virulent EHV-1. In contrast to control animals, thenumber of clinical signs of EHV-1 respiratory disease, the severity ofthe clinical signs and the number of vaccinates exhibiting clinicalsigns were significantly reduced in vaccinated horses. Both theintramuscular and intramuscular/intranasal routes of vaccination weresimilar in the reduction in clinical signs of respiratory disease due toEHV-1 infection. Reduction in clinical disease in the vaccinates issupported by data that fewer intramuscular and intramuscular/intranasalvaccinated horses shed virus post EHV-1 challenge and shed virus forshorter periods of time.

The results of the study demonstrated that the vaccine containinginactivated EHV-1 generated VN antibody was immunogenic for protectionof horses against respiratory disease caused by EHV-1 when administeredby either the intramuscular or intramuscular/intranasal routes.

EXAMPLE 7 Production of Immunogenic Compositions Containing InactivatedEHV-1 and EHV-4 Strains

To produce the combined vaccine, master seed cultures of EHV-1 and EHV-4were first produced. From these master seeds, separate cultures of EHV-1and EHV-4 were grown and then inactivated. The inactivated viruscultures are then mixed with adjuvant to produce the combined vaccine.The method used to produce the combined inactivated EHV-1/EHV-4 vaccineis described below.

Fluids containing inactivated EHV-1 KyA from the master seed virusculture fluid designated EHV-1 KyA, MSV Lot 001-dil were producedaccording to the procedure described in Example 1.

To create the master seed virus of equine herpesvirus type 4 (EHV-4),personnel from Boehringer Ingelheim Vetmedica, Inc., isolated virus froma horse infected with rhinitis. The isolated virus was passaged fivetimes on Vero A 139 cells and three times on EVero cells. The thirdpassage was used as a master seed virus and designated EHV-4, MSV Lot001-dil.

Cultures of EHV-4 were produced by infecting EVero cells with seed viruscontained in a minimum essential medium (MEM) having 0 to 5% serum. Thecultures were then incubated at 36° C.±2° C. for 24 to 120 hours inglass roller bottles or on microcarrier beads. During incubation, thecultures were checked for EHV induced cytopathic effects (CPE) to ensurethe purity of the EHV strain. If atypical CPE were present or anymacroscopic or microscopic evidence of contamination existed, theculture was discarded. Pure virus cultures were aseptically harvestedinto sterile glass carboys, sterile plastic carboys, or sterilestainless steel tanks and were clarified by filtration through filtersof 8 microns or greater. After being harvested, the virus culture wasinactivated in order to produce a killed vaccine using the proceduredescribed for EHV-1 in Example 1.

After inactivation, the cultures were tested for CPE typical of EHV toensure inactivation of the virus using the procedure described inExample 5. This task was accomplished by passing the BEI treated viralfluids over EVero cells and checking the EVero cells for any viralinfection. After a satisfactory inactivation test showing no viralinfection, the BEI was neutralized by adding a cold (4° C.±2° C.)solution of 1.0 M sodium thiosulfate to give a final concentration of 6mM.

Following the inactivation and testing of the EHV-1 and EHV-4 cultures,the cultures were blended with an adjuvant to form the final product,the combined inactivated EHV-1/EHV-4 vaccine. This final productcontained EHV-1 fluids, EHV-4 fluids, adjuvant stock solution (0.5%Carbopol® 971), and saline solution in a ratio of 3.75:3.00:12.00:41.19.Typically, a batch contained 3,750 mL of EHV-1, 3,000 mL of EHV-4,12,000 mL of adjuvant solution, and 41,190 mL of saline, yielding atotal volume of 60 L of a bulk serial.

EXAMPLE 8 Inoculation of Horses with the Combined EHV-1 and EHV-4Vaccine and Subsequent Challenge with Virulent EHV-4

An experiment was performed to demonstrate the immunogenicity of thecombination inactivated EHV-1/EHV-4 vaccine. Six groups of male andfemale horses that ranged in age from four to seven months and werevirus neutral to EHV-1 and EHV-4 were used in the experiment. Asillustrated in the Table VIII-1 below, three groups were challenged withvirulent EHV-1 while three groups were challenged with virulent EHV-4.Of the horses challenged with the EHV-1, one group was vaccinated withthree intramuscular (“IM”) injections, one group was vaccinated with twointramuscular injections followed by one intranasal (“IN”)administration, and one group was not vaccinated. Similarly, of thehorses challenged with the EHV-4, one group was vaccinated entirely withintramuscular injections, one group was vaccinated with twointramuscular injections followed by one intranasal administration, andone group was not vaccinated.

The horses were vaccinated at three week intervals with 2 ml doses ofthe combined vaccine having a RPV of 1.0 per dose of inactivated EHV-1and 1.0 per dose of inactivated EHV-4. During the trial, the horses weremonitored for signs of respiratory disease. No clinical signs ofrespiratory disease were observed in any of the horses during thevaccination period. TABLE VIII-1 Summary of Combined EHV-1/EHV-4 VaccineTest Group No. animals Vaccination Method Challenge Group IV-1 10 IM,IM, IM EHV-1 Group IV-2 10 IM, IM, IN EHV-1 Group IV-3 10 None (controlgroup) EHV-1 Group IV-4 10 IM, IM, IM EHV-4 Group IV-5 10 IM, IM, INEHV-4 Group IV-6 10 None (control group) EHV-4

Animals were challenged at 3 weeks after vaccination with eithervirulent EHV-1 or virulent EHV-4 at a target dose of 5.0 TCID₅₀ Log₁₀/2ml. Specifically, EHV-1 Kentucky D strain (approximate dose of 4.5TCID₅₀ Log₁₀/2 ml) and EHV-4 405 strain (approximate dose of 4.0 TCID₅₀Log₁₀/2 ml) were used as the challenge viruses and were administeredintranasally in 2 ml doses (administered as 1 ml dose per nostril). Theprotection of the vaccine was measured by monitoring the horses forclinical signs of respiratory disease, such as pyrexia, nasal discharge,conjunctivitis, coughing, dyspnea, and depression, and measuring theseverity of the disease. Additionally, blood and nasal samples weretaken to measure the amount of virus shedding.

Post virus challenge, all groups of vaccinated horses challenged witheither EHV-1 or EHV-4 showed a significant reduction in signs ofrespiratory disease, with little difference displayed between theparenteral vaccinate groups and the parenteral/intranasal vaccinategroups. Specifically, the vaccinated animals experienced a significantdecrease in nasal discharge, conjunctivitis, coughing, dyspnea, anddepression. These results demonstrate that the EHV-1 and EHV-4 antigenscontained in the combined inactivated EHV-1/EHV-4 vaccine areimmunogenic when administered by either the parenteral orparenteral/intranasal route.

In addition to the vaccinated horses showing a reduction in respiratorydisease, the vaccinated horses also displayed a reduction in virusshedding. Of the vaccinated horses challenged with EHV-1, virus sheddingat 1 to 2.5 log₁₀ TCID₅₀ /ml for one to three days was only observedwith only 30% of the parenteral vaccinate group and 40% of theparenteral/intranasal group. In contrast, 90% of the non-vaccinatedcontrol group shed the virus at 2.5 to 3.75 log₁₀ TCID₅₀ /ml for two toseven days. Similarly, of the vaccinated horses challenged with thevirulent EHV-4 strain, virus shedding at 1 to 2.5 log₁₀ TCID₅₀ /ml forone to three days was only observed with 40% of the parenteral vaccinategroup and 50% of the parenteral/intranasal group. Again, thenon-vaccinated horses exhibited much greater virus shedding with 100% ofthe non-vaccinated control group shedding the virus at 1 to 5.25 log₁₀TCID₅₀ /ml for two to seven days. These statistics are evidence of asignificant reduction in the amount and number of days the vaccinatedhorses shed the virus as compared to the non-vaccinated horses. Suchevidence establishes that the EHV-1 and EHV-4 antigens contained in thecombined inactivated EHV-1/EHV-4 vaccine are immunogenic whenadministered by either the parenteral or parenteral/intranasal route.

EXAMPLE 9 Inoculation of Horses with the Combined EHV-1/EHV-4/EquineInfluenza Vaccine and Subsequent Challenge with Virulent EquineInfluenza Virus

An experiment was conducted to evaluate the immunogenicity of the EIVvaccine fractions by evaluation of the serological response to the EIVA1 and A2 subgroups in the host animal. The study was also designed todemonstrate noninterference of EIV and EHV components in a combinationrhinopneumonitis-influenza vaccine, killed virus by serologicalevaluation in the host animal. The third objective was to demonstratethat the EIV A1 subgroup and EIV A2 subgroups in the influenza vaccineand the rhinopneumonitis-influenza combination vaccines are immunogenicwhen administered by the parenteral and parenteral/intranasal routes.

Study Design

Vaccine A was administered to horses by two vaccination regimens. Onevaccination regimen was three intramuscular vaccinations at three weekintervals. The other regimen was two intramuscular vaccinations at threeweeks apart and the third vaccination by the intranasal route threeweeks later. Each vaccination regimen group contained 20 horses. A groupof five horses served as nonvaccinated controls. Blood samples and nasalwashings were taken before and at selected times post vaccination forevaluation of the serological response to each of the three EW vaccinestrains. Blood and nasal washings were collected from the horses atperiodic intervals. Horses were observed for general health and anyabnormal behavior or health conditions during the 63 day experimentalperiod.

Horses

Healthy male and female horses that ranged in age of seven to ninemonths were obtained from selected sources. Horses were identified bymicrochip numbers and were randomized into groups by drawing theidentification numbers of the horses from a sack. During theexperimental period, horses were maintained in open pens and fed freechoice dairy quality alfalfa hay, Sweet 14 dietary supplement, equineBio-mineral, and water ad libum. Horses were observed for general healthand any abnormal behavior during the experimental period. No abnormalbehavior or adverse health conditions were observed in any of the horsepost vaccination and no adverse injections site reactions were observedin any horse post vaccination. At the time of the first vaccination,horses vaccinated with Vaccine A had hemagglutination inhibition (HAI)antibody titers of ≦10 to EIV A1 and A2 subgroups.

Vaccine

The vaccine included EIV killed virus that contained EIV subgroup A1 andantigenically relevant EIV A2 subgroup strains. Because, A2 subgroups inNorth America differ from A2 subgroups in Europe, the vaccine containedstrains, designated Kentucky/95 and Newmarket/2/93, that arerepresentative of the A2 subgroups in North America and Europe,respectively. EIV and EHV virus fluids for use in the vaccine wereproduced according to the procedures described in Example 7. Virusfluids were at the fifth passage from master seed virus and wereproduced on cells at the 20^(th) passage from master cell stock. VaccineA was formulated to contain 64 HA units of the EIV A1 subgroup, 128 HAunits each of the two EIV A2 subgroups, and ≧3.0 relative potency (RP)units of EHV-1 and ≧3.0 relative potency (RP) units of EHV-4 per two mldose. The vaccine was labeled as EIV/EHV Imm/Intfr, 623-0856-98E-107,Vaccine A Lot 001, 12-15-98.

Potency determination of EIV and EHV fractions of Vaccine A

The potency of the EIV fractions in the vaccine were determined by theNational Veterinary Services Laboratories Testing Protocol, SupplementalAssay Method for Conducting the Hemagglutination Inhibition Assay forEquine Influenza Antibody (MVSAM0124.01, dated Oct. 2, 1998). Relativepotency values of the EHV-1 and EHV-4 fractions in Vaccine A weredetermine by the EHV ELISA Potency Release Assay described in Example 4.The potency of Vaccine A was satisfactory for the EIV fractions. Ten often guinea pigs had HAI antibody titers of 80 or greater for the A1subgroup and 10 of 10 guinea pigs had HAI antibody titers of 40 orgreater for each A2 subgroup in the vaccine. The relative potency valuewas 4.73 and 3.31 for the EHV-1 and EHV-4 fractions, respectively.

Serum HAI antibody titers to EIV subgroups A1 and A2 post vaccination

The HAI antibody titers in horses after vaccination with Vaccine A weredetermined. All vaccinates were seronegative to all three EIV strains atthe time of the first vaccination. At the prevaccination time period,two of the nonvaccinated control horses had HAI antibody titers of 20 tothe EIV A1 and the other three control horses were seronegative to theEIV A1. All five nonvaccinated control horses were seronegative to thetwo EIV A2. During the experimental period, nonvaccinated control horsesremained seronegative to the two EIV subgroup A2 and did not show morethan a two-fold variation in HAI antibody titer to the EIV subgroup A1.There was no indication of exposure to field EIV during the experimentalperiod. At three weeks post first vaccination, the majority of horsesshowed a serological response to the EIV A1 subgroup and to the EIV A2NM subgroup. After one vaccination, only four horses had a serologicalresponse to the EIV A2 K subgroup. The number of horses with aserological response to the EIV A2 K subgroup increased after the secondvaccination. After the third vaccination, 19 of 20 horses (95%) thatreceived three intramuscular vaccinations had HAI antibody titers of 40or greater to the EIV A1 subgroup. After three intramuscular injections,18 of 20 (90%) and 20 of 20 (100%) of the horses had HAI antibody titersof 20 or greater to the EIV A2 K and EIV A2 NM subgroups, respectively.Similarly, 17 of 20 horses (85%) that received two intramuscularvaccinations and one intranasal vaccination had HAI antibody titers of40 or greater to the EIV A1 subgroup. Eighteen of 20 (90%) and 20 of 20(100%) horses had HAI antibody titers of 20 or greater to the EIV A2 Kand EIV A2 NM subgroups, respectively, after two intramuscular and oneintranasal vaccination. Geometric mean antibody titers after the thirdvaccination were 45, 35 and 61 for the EIV A1, A2 K, and A2 NMsubgroups, respectively, in horses that received three intramuscularinjections. Geometric mean antibody titers in horses that received twointramuscular and one intranasal vaccination were 39, 24, and 51 for theEIV A1, A2 K, and A2 NM subgroups, respectively.

Mucosal HAI Antibody Titers to EIV Subgroups A1 and A2 Post Vaccination

The HAI antibody titers in nasal samples of horses after vaccinationwith Vaccine A were determined. At the time of the first vaccination,AHI antibody to the two EIV A2 subgroups were not detected in any nasalsamples from the horses. Hemagglutination inhibition titers to the EIVsubgroup A1 were detected in some of the vaccinates and nonvaccinatedcontrol horses at the first vaccination. After the first and secondvaccination, levels of AHI antibody in nasal secretions were variable.After the final intramuscular or intranasal vaccination, HAI antibodylevels were highest to the EIV A1 subgroup compared to the two A2subgroups. There was little to no HAI antibody to the EIV A2 K subgroupin nasal samples from horses post third vaccination by eitherintramuscular or intranasal routs. Interestingly, HAI antibody levels tothe EIV A1 subgroup and A2 NM subgroup were lower in horses thatreceived the third vaccination by the intranasal route.

DISCUSSION

One purpose of this study was to demonstrate the immunogenicity of theEIV A1 and A2 fractions in the vaccine, when administered by eithervaccination regimen. Immunogenicity was assessed by determination of theserum HAI antibody response to the three EIV strains after the finalvaccination. Results demonstrated that greater than 80% of the horses inboth vaccination regimen groups had serum HAI antibody titers of 40 orgreater to the EIV A1 subgroup after the final vaccination and greaterthan 80% of the horses in both vaccination regimen groups had serum HAIantibody titers of 20 or greater to both EIV A2 subgroups after thefinal vaccination. HAI antibody levels to the EIV A1 and A2 subgroupswere also determined in nasal samples at selected times postvaccination. Mucosal HAI antibody titers were lower than serum HAIantibody titers in nasal samples from horses in both vaccinationregimens and, in contrast to serum HAI titers, mucosal HAI titersincreased very little after each vaccination. It is possible that thehemagglutination inhibition assay does not detect the isotype ofantibody that is most prevalent in nasal samples. The experimentsdemonstrates that the EIV A1 subgroup and A2 subgroups in the influenzavaccine, killed virus and the rhinopneumonitis-influenza vaccine, killedvirus, were immunogenic when administered by both the parenteral andparenteral/intranasal routes. In particular, the study demonstrated thatthe EIV NM/77 A1 subgroup and the K95 A2 subgroup and NM/2/93 A2subgroups were immunogenic.

Another purpose of the study was to demonstrate non-interference of theEIV and EHV vaccine fractions with each other. Vaccine A used in thestudy was formulated with the minimum release dose of 64 and 128 HAunits of the EIV A1 and A2 fractions, respectively, and formulated witha relative potency value of three fold or greater for the EHV-1 andEHV-4 fractions. Results of the study demonstrated that a vaccinecontaining the minimum antigen dose of the EIV fractions and more thanthe minimum EHV antigen dose is capable of generating a satisfactoryserological response to the EIV A1 and A2 subgroups in the host animal.Thus, the EHV-1 and EHV-4 fractions did not interfere with theimmunogenicity of the EIV vaccine fractions. Likewise, the EHV fractionsdid not result in an unsatisfactory potency test in the guinea pigmodel.

The invention has been described with reference to various specific andillustrative embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention. While variousembodiments are discussed in some detail herein, it should beappreciated that the present invention provides inventive concepts whichcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the present immunogenic compositions and are not meant tolimit the scope of the invention. Various modifications and combinationsof the illustrative embodiments, as well as other embodiments of theinvention, will be apparent to persons skilled in the art upon referenceto the disclosure herein. TABLE IV-1 PLATE TEMPLATE FOR EHV ELISA 1 2 34 5 6 7 8 9 10 11 12 A Ref Ref Test 1 Test 1 Test 2 Test 2 Test 3 Test 3Test 4 Test 4 Vaccine Vaccine Vaccine Vaccine Vaccine Vaccine VaccineVaccine Vaccine Vaccine B Blank Undilute Undilute Undilute UndiluteUndilute Undilute Undilute Undilute Undilute Undilute External ReferenceC Blank 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:2 External Reference DBlank 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 External Reference E Blank1:8 1:8 1:8 1:8 1:8 1:8 1:8 1:8 1:8 1:8 Negative Control F 1:16 1:161:16 1:16 1:16 1:16 1:16 1:16 1:16 1:16 Negative Control G 1:32 1:321:32 1:32 1:32 1:32 1:32 1:32 1:32 1:32 Negative Control H

1. A vaccine for protecting a horse against diseases associated withEHV-1, EHV-4 or a combination thereof comprising: chemically inactivatedEHV-1 KyA virus; and an adjuvant.
 2. A method of producing an equineherpesvirus vaccine comprising: (a) inoculating simian cells with anEHV-1 KyA virus; (b) incubating the inoculated simian cells; (c)harvesting EHV-1 KyA virus from the incubated cells; and (d) treatingthe harvested cells with a chemical inactivating agent which includes acompound selected from the group consisting of ethylenimine, binaryethylenimine, acetylethylenimine or a mixture thereof to forminactivated EHV-1 KyA virus.
 3. A kit for detecting EHV-1 and EHV-4 in atest sample comprising: (a) a first monoclonal antibody thatimmunoreacts with EHV-1 to form a first complex; (b) a second monoclonalantibody that immunoreacts with EHV-4 to form a second complex; and (c)at least one detectable label for use in detecting the first complex,the second complex, or both complexes.